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. 2014 Dec 23;111(51):18321-6.
doi: 10.1073/pnas.1406199111. Epub 2014 Dec 8.

Microbiota Organization Is a Distinct Feature of Proximal Colorectal Cancers

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Free PMC article

Microbiota Organization Is a Distinct Feature of Proximal Colorectal Cancers

Christine M Dejea et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Environmental factors clearly affect colorectal cancer (CRC) incidence, but the mechanisms through which these factors function are unknown. One prime candidate is an altered colonic microbiota. Here we show that the mucosal microbiota organization is a critical factor associated with a subset of CRC. We identified invasive polymicrobial bacterial biofilms (bacterial aggregates), structures previously associated with nonmalignant intestinal pathology, nearly universally (89%) on right-sided tumors (13 of 15 CRCs, 4 of 4 adenomas) but on only 12% of left-sided tumors (2 of 15 CRCs, 0 of 2 adenomas). Surprisingly, patients with biofilm-positive tumors, whether cancers or adenomas, all had biofilms on their tumor-free mucosa far distant from their tumors. Bacterial biofilms were associated with diminished colonic epithelial cell E-cadherin and enhanced epithelial cell IL-6 and Stat3 activation, as well as increased crypt epithelial cell proliferation in normal colon mucosa. High-throughput sequencing revealed no consistent bacterial genus associated with tumors, regardless of biofilm status. However, principal coordinates analysis revealed that biofilm communities on paired normal mucosa, distant from the tumor itself, cluster with tumor microbiomes as opposed to biofilm-negative normal mucosa bacterial communities also from the tumor host. Colon mucosal biofilm detection may predict increased risk for development of sporadic CRC.

Keywords: adenoma; bacterial communities; biofilm; colorectal cancer; microbiome.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Detection and quantification of bacterial biofilms on colon tumors. (A) FISH of all bacteria (red) on cancers (Top), paired normal tissue from patients with CRC (Middle), and colonoscopy biopsies from healthy individuals without CRC (Bottom). All were counterstained with the nuclear stain, DAPI (blue). The top white brackets demarcate the mucus layer and the bottom white brackets denote the cytoplasm separating the nucleus (blue) of the colorectal epithelium from the mucus layer. PAS stains (SI Appendix, Fig. S2) further delineate the mucus layer on these samples. (Insets) Closeup (100×) showing close contact between bacteria and epithelial cells in patient A. The pale, nonpunctate red staining of the mucus layer in patients without biofilms (patient B) represents nonspecific binding to the mucus layer, which is easily demarcated from the bright red punctate staining of the bacteria infiltrating the mucus layer in patients with biofilms. (Scale bars, 50 µm.) (B) Biofilm depth and density measurements from right CRCs/surgical normal pairs (n = 15), right adenomas/surgical normal pairs (n = 4), left CRCs/surgical normal pairs (n = 15), left adenomas/surgical normal pairs (n = 2), and right/left paired normal colonoscopy biopsies from healthy individuals without CRC (n = 60). Data displayed as bar and whisker graphs, where line designates the median, boxes the 25/75th percentile, and whiskers the 95th percentile. (C) Geographical distribution of tumors (CRC, n = 30, and adenomas, n = 6) with biofilm designation. (D) SEM images. (Left) Biofilm on a right colon cancer dominated by filamentous bacteria. (Center) Biofilm-negative left colon cancer where no bacteria are visualized. (Right) Image of bacterial contact with host epithelium (white arrow) on biofilm-covered right colon adenoma. Mixed bacterial morphology (*rods and cocci) is seen. (Scale bars, 2 µm.)
Fig. 2.
Fig. 2.
FISH and sequencing analysis of tissue reveal invasive polymicrobial biofilms and transitioning microbial populations. (A–C) Multiprobe spectral images of FISH-targeted bacterial groups (40×). Bacteroidetes (green), Lachnospiraceae (magenta), Fusobacteria (cyan), Enterobacteriaceae (orange), Bacteroides fragilis (red) are represented within the biofilms, and tissue autofluorescence is white. (A) Multigroup bacterial biofilm with invasion of cancer tissue (white arrows). Dotted white line depicts margin between bacterial biofilm and cancer tissue in lower right portion of image. Right cancer with a Fusobacteria dominant polymicrobial biofilm (by sequencing analysis; see text) also containing Bacteroidetes, Lachnospiraceae, and Enterobacteriaceae. Dominant group in left cancer is Bacteroidetes. B. fragilis, Lachnospiraceae, and Fusobacteria are also present. Cancer-invading bacteria represent a subset of biofilm community members. (B) Bacterial biofilms on paired surgical normal tissue from CRC patients, comprising Lachnospiraceae, Bacteroidetes, and Enterobacteriaceae. (C) Thin bacterial biofilms detected on right (Bacteroidetes, Lachnospiraceae, and Enterobacteriaceae) and left (Bacteroidetes and Lachnospiraceae) normal colonoscopy biopsies from two different individuals without CRC. (D, Left) All bacteria FISH (red) with DAPI nuclear counterstain (blue) of surgically resected normal tissue covered by a biofilm (20×) from a patient with CRC. White arrows mark two sites of biofilm infiltration of the epithelial tissue (20×). (D, Right) Confocal z-stack of tissue bacterial (red) invasion (40×) denoted by white box in D, Left. Disordered epithelial cells and leukocytes are visible at the infiltrated sites, whereas surrounding epithelial cells are intact and ordered as seen in D, Left. (Scale bars, 50 µm in A–D.) (E) Histogram of bacterial classes represented on biofilm-positive and -negative samples as defined by sequence analysis. Tumor denotes 23 CRCs and two adenomas. (F) PCoA plot (based on unweighted UniFrac distances) displaying mucosa community structure of all samples (each point reflects an individual sample). Normal colonoscopy biopsies from healthy individuals without CRC (n = 21, red) and surgically resected normal tissues without a biofilm from patients with CRC (n = 12, orange) transition to normal tissues with a biofilm from patients with CRC (n = 13, green) that cluster more closely to biofilm-positive adenomas (n = 2, dark blue squares) and CRCs with (n = 12, dark blue) and without (n = 11, light blue) biofilms.
Fig. 3.
Fig. 3.
Biofilms are associated with changes in E-cadherin, IL-6, and Stat3 activation. (A and B) Evaluation of E-cadherin and IL-6 by immunofluorescence (green) and activated Stat3 (pStat3, brown nuclei) by immunohistochemistry. Blue, nuclear DAPI counterstain; red, smooth muscle antigen. [Scale bars, 100 μm (E-cadherin) and 50 μm (IL-6, pStat3).] (A) Normal colonic tissues associated with a biofilm from patients with CRC (Left), obtained during surgery, display diminished crypt colonic epithelial cell E-cadherin (white arrows, n = 7 biofilm-positive or -negative tissues) and increased epithelial cell IL-6 (white arrows, n = 13 biofilm-positive or -negative tissues), as well as epithelial cell pStat3 (black arrows, n = 16 biofilm-positive and n = 12 biofilm-negative tissues). Normal colonic tissues without a biofilm from patients with CRC (Right), likewise obtained during surgery, display intact E-cadherin. IL-6 and pStat3 are detected in the lamina propria. (B) Biofilm-positive colonoscopy biopsies from subjects without CRC (Left) display epithelial cell E-cadherin redistribution (Inset) and increased tissue IL-6, whereas biofilm-negative colonoscopy biopsies (Right) display intact E-cadherin and modest lamina propria IL-6 expression. pStat3 is observed in the lamina propria immune cells in both biofilm-positive and biofilm-negative colonoscopy biopsies. (C–F) Quantification of crypt cell E-cadherin (fluorescence intensity), epithelial cell IL-6 [fluorescence intensity and isolated colonic epithelial cells (CEC) by ELISA], and epithelial cell pStat3 (immunohistochemistry) from A are shown in C–F, respectively. Data displayed as bar and whisker graphs, where line designates the median, boxes the 25/75th percentile, and whiskers the 95th percentile (C and D) or mean ± SD (E and F). Details in SI Appendix, Materials and Methods. SI Appendix, Figs. S10 and S11 show additional E-cadherin, IL-6, and pStat3 quantification from A and B.
Fig. 4.
Fig. 4.
(A and B) Scoring of Ki67-positive cells from the base of the crypt to the luminal surface. Normal tissues from patients with CRC obtained at surgery (A) with (n = 17) and without (n = 18) a biofilm, as well as normal mucosa from healthy subjects obtained via colonoscopy (B) with (n = 7) and without (n = 10) a biofilm, displayed increased proliferation in a biofilm setting. Data displayed as mean ± SEM in groups based on distance from crypt base (<15 cells, 15–30 cells, >30 cells).

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